Mature articular chondrocytes are maintained in a highly differentiated state at the joint through the action of local growth factors, transcription factors, and the interaction with extracellular matrix components. Growth factors like transforming growth factor (TGF)-beta and the chondrogenic transcription factor Sox9 are associated with the maintenance of mature articular cartilage through their action on chondrocyte differentiation status. However, the molecular mechanisms by which TGF-beta acts on Sox9 in chondrocytes are not yet fully understood. Preliminary studies indicate that TGF-2 enhances Sox9 protein expression independently of effects on mRNA expression. Furthermore, the data support a role for TGF-beta-dependent induction of Sox9 protein sumoylation, a protein modification that can change protein stability, activity and cellular localization. This proposal is designed to characterize the role of TGF-2-mediated sumoylation on Sox9 protein levels and activity as well as its role in chondrocyte differentiation. We hypothesize that TGF-beta maintains the differentiated chondrocyte phenotype in permanent cartilages, like articular cartilage, by regulating Sox9 levels and activity via protein sumoylation. Three specific aims have been developed to address our hypothesis: 1) to determine which sites on Sox9 are sumoylated in response to TGF-beta and determine the role of sumoylation in TGF-beta-mediated Sox9 levels, localization, and activity;2) to determine the mechanism of TGF-beta-mediated sumoylation of Sox9;and 3) to determine the role of sumoylation on TGF-beta-mediated chondrocyte differentiation. The information derived from these studies will provide critical insight into the molecular mechanisms of cartilage maintenance through control of chondrocyte differentiation. The proposed studies are designed to identify mechanisms that can be targeted for future interventions in osteoarthritis to prevent cartilage degeneration or improve cartilage maintenance. PUBLIC HEALTH RELEVANCE: It has been shown that the loss of Sox9 expression correlates with the progression of osteoarthritis (OA), the most common form of arthritis and the primary cause of disability in the US. Our preliminary data suggests that transforming growth factor beta (TGF-beta) signaling may play a critical role in cartilage maintenance and repair by increasing Sox9 levels in cartilage cells via a post-translational modification called sumoylation. Studies aimed at understanding how the Sox9 protein is modified in its expression and activity in response to TGF-beta will enhance our current knowledge of how cartilage is maintained and provide new therapeutic targets for cartilage regeneration in OA.